U.S. patent application number 10/492980 was filed with the patent office on 2004-12-30 for amperometer sensor.
Invention is credited to Lau, King Tong, Murphy, Lindy, Slater, Jonathan M.
Application Number | 20040265940 10/492980 |
Document ID | / |
Family ID | 9924134 |
Filed Date | 2004-12-30 |
United States Patent
Application |
20040265940 |
Kind Code |
A1 |
Slater, Jonathan M ; et
al. |
December 30, 2004 |
Amperometer sensor
Abstract
An amperometric sensor suitable for determining the
concentration of analyte in a sample, said sensor comprising an
oxidase enzyme and a substituted 1,4-benzoquinone compound which,
in oxidized form, functions as a mediator specific to reduced
enzyme and which has a lower oxidation potential than unsubstituted
1,4-benzoquinone.
Inventors: |
Slater, Jonathan M; (London,
GB) ; Murphy, Lindy; (London, GB) ; Lau, King
Tong; (Dublin, IE) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
1100 N GLEBE ROAD
8TH FLOOR
ARLINGTON
VA
22201-4714
US
|
Family ID: |
9924134 |
Appl. No.: |
10/492980 |
Filed: |
May 5, 2004 |
PCT Filed: |
September 18, 2002 |
PCT NO: |
PCT/GB02/04268 |
Current U.S.
Class: |
435/14 |
Current CPC
Class: |
C12Q 1/004 20130101;
C12Q 1/006 20130101 |
Class at
Publication: |
435/014 |
International
Class: |
C12Q 001/54 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 18, 2001 |
GB |
0125094.3 |
Claims
1. An amperometric sensor suitable for determining the
concentration of analyte in a sample, said sensor comprising an
oxidase enzyme and a substituted 1,4-benzoquinone compound which,
in oxidized form, functions as a mediator selective for reduced
enzyme and has an oxidation potential lower than that of
unsubstituted 1,4-benzoquinone.
2. A sensor according to claim 1, wherein the analyte is glucose
and the enzyme is glucose oxidase.
3. A sensor according to claim 1, wherein the substituted
1,4-benzoquinone compound is of formula (I): 3wherein R.sup.1,
R.sup.2, R.sup.3 and R.sup.4 are the same or different and at least
one of R.sup.1, R.sup.2, R.sup.3 and R.sup.4 is an alkyl group or a
phenyl group.
4. A sensor according to claim 3, wherein the alkyl groups have
from 1 to 3 carbon atoms.
5. A sensor according to claim 4, wherein the 1,4-benzoquinone
compound is chosen from 2,6-dimethyl-1,4-benzoquinone,
tetramethyl-1,4-benzoquinone, methyl-1,4-benzoquinone,
2,5-dimethyl-1,4-benzoquinone and phenyl-1,4-benzoquinone.
6. A sensor according to claim 1, wherein the 1,4-benzoquinone
compound is of formula (I) wherein R.sup.1, R.sup.2, R.sup.3 and
R.sup.4 are the same or different and at least one of R.sup.1,
R.sup.2, R.sup.3 and R.sup.4 is an alkoxyl group or a hydroxyalkyl
group.
7. A sensor according to claim 6 wherein the alkoxyl group or
hydroxyalkyl group have from 1 to 3 carbon atoms.
8. A sensor according to claim 6, wherein the benzoquinone compound
is chosen from
2,6-dimethoxy-1,4-benzoquinone,2,3-dimethoxy-5-methyl-1,4-ben-
zoquinone and 2-hydroxymethyl-6-methoxy-1,4-benzoquinone.
9. A cartridge for an amperometric sensor suitable for measuring
analyte in a sample, which cartridge comprises an enzyme and a
substituted benzoquinone compound as defined in claim 1.
10. Use of an amperometric sensor as claimed in any one claim 1 for
determining the concentration of an analyte in a sample, wherein
the enzyme of the sensor reacts with the analyte to produce reduced
enzyme which is then regenerated by the mediator.
Description
[0001] In general terms the present invention relates to the
determination of the concentration of an analyte in a sample. More
specifically, the invention relates to an amperometric sensor, to
its use, to cartridges for the sensor and to redox mediator
compounds for use in the sensor.
[0002] A number of electrochemical sensors (or biosensors) have
been proposed previously. For example, U.S. Pat. No. 5,288,636
describes a sensor useful for determining glucose concentration in
a sample and relies on the reaction between the enzyme glucose
oxidase and glucose with the mediator potassium ferricyanide to
produce a ferrocyanide which is then electro-oxidised to produce a
measurable current that is representative of the concentration of
glucose present.
[0003] The reactions involved can be summarised as follows:
[0004] 1. GOD.sub.ox+glucose.fwdarw.GOD.sub.RED+gluconolactone
[0005] 2. GOD.sub.RED+M.sub.OX.fwdarw.GOD.sub.OX+M.sub.RED
[0006] 3. M.sub.RED.fwdarw.M.sub.OX+e.sup.-[Signal]
[0007] GOD.sub.OX.fwdarw.oxidised form of glucose oxidase
[0008] GOD.sub.RED--reduced form of glucose oxidase
[0009] M.sub.OX--oxidised form of mediator (ferricyanide)
[0010] M.sub.RED--reduced form of mediator (ferrocyanide)
[0011] In step 1 the enzyme oxidises the glucose and is itself
reduced. In step 2 the reduced form of the enzyme reacts with the
oxidised form of the mediator to produce the reduced form of the
mediator and to regenerate the oxidized form of the enzyme. In step
3 the oxidised form of the mediator is regenerated by
electro-oxidation. A measurable current/signal is generated. Thus,
this type of sensor depends on reaction between the mediator and
enzyme.
[0012] The use of a quinone-hydroquinone mediator system with
glucose oxidase is known (Stoytcheva et al, Analytica Chimica Acta,
315 (1995) pp 101-107). However, unsubstituted benzoquinone has the
disadvantage of having a relatively high oxidation potential of
approximately +400 mV (versus SCE).
[0013] U.S. Pat. No. 4,711,245 also describes a sensor for
determining glucose concentration. The sensor relies on a reaction
involving glucose, the enzyme glucose oxidase and the oxidised form
of a substituted ferrocene. The ferrocene is reduced and then
reoxidised to produce an easily measurable current.
[0014] There are various advantages associated with mediated
sensors. Firstly, the kinetics for the oxidation of reduced enzyme
can be faster with the mediator than with the natural electron
acceptor oxygen. This can result in the sensor response being
independent of oxygen tension. Secondly, in known sensors a
potential is applied between electrodes in order to oxidize the
reduced form of the mediator. The potentials sufficient to achieve
this can be lower than for known sensors based on oxidation of
hydrogen peroxide. This can result in a reduced oxidation of
interferants present in the system, for example, ascorbate, urate
and paracetamol.
[0015] The solubility of the mediator is an important factor in
obtaining a measurable sensor response. The use of mediated sensors
in flow systems can be limited by the solubility of the mediator. A
readily soluble mediator can be lost rapidly to the carrier
solution, resulting in severely reduced operating lifetime of the
sensor. Alternatively, where the sensor is used in static solution
and possibly with minimal sample volume, such as an electrochemical
blood glucose stick, the use of a mediated sensor is enhanced by
the solubility of the mediator. A poorly soluble mediator will
limit the mediated enzyme reaction, resulting in poor sensitivity
of the sensor.
[0016] The present invention provides mediators suitable for use in
flowing streams by use of a sensor reliant on relatively insoluble
mediator. The present invention also provides mediators suitable
for use in static solution by use of a sensor reliant on relatively
soluble mediator.
[0017] Accordingly, the present invention provides an amperometric
sensor suitable for determining the concentration of analyte in a
sample, said sensor comprising an oxidase enzyme and a substituted
1,4-benzoquinone compound which, in oxidized form, functions as a
mediator selective for reduced enzyme and which has an oxidation
potential lower than that of unsubstituted 1,4-benzoquinone.
[0018] The enzyme is an oxidase type enzyme. For example, in a
sensor for determining the concentration of glucose in a sample,
the enzyme may be glucose oxidase. The reaction between the enzyme
and the analyte yields reduced glucose oxidase, and the
concentration of the reduced glucose oxidase can be determined by
using the sensor response and correlating this to a corresponding
glucose concentration.
[0019] Other analytes which may be determined using the sensor of
the present invention include cholesterol, pyruvate, bilirubin,
alcohol, lactate, sarcosine, glycerol, creatinine, triglycerides
and cholesterol; U.S. Pat. No. 5,288,636 gives details of some of
the relevant enzymes. These analytes may be measured if one or more
suitable enzyme(s) and mediator are included in the sensor. The
sensors are constructed so that the final enzyme reaction results
in the formation of reduced enzyme, which can be detected by the
substituted benzoquinone mediator.
[0020] Herein the term "mediator" as used herein, means a compound
which is capable of undergoing an electrochemical, reversible
oxidation-reduction reaction.
[0021] The mediator used in the present invention is a substituted
1,4-benzoquinone compound which in oxidized form is selective for
the redox site of reduced enzyme, i.e. which is reduced on reaction
with reduced enzyme. The mediator has satisfactory solubility in
water and common organic solvents, and has a lower redox potential
than the corresponding unsubstituted benzoquinone. Examples of
suitable compounds include those of the following formulae (I):
1
[0022] wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are the same
or different and at least one of R.sup.1, R.sup.2, R.sup.3 and
R.sup.4 is an alkyl group preferably C.sub.1-3 alkyl or a phenyl
group. Alternatively the groups R.sup.1, R.sup.2, R.sup.3 and
R.sup.4 may be chosen from an alkoxyl group or a hydroxyalkyl
group, preferably C.sub.1-3 alkoxyl or hydroxy C.sub.1-3 alkyl.
Preferably the substituents are chosen from methyl, methoxy and
hydroxymethyl groups.
[0023] Suitable 1,4-benzoquinone mediators for use in the sensor of
the invention include:
[0024] methyl-1,4-benzoquinone
[0025] ethyl-1,4-benzoquinone
[0026] propyl-1,4-benzoquione
[0027] 2,5-dimethyl-1,4-benzoquinone
[0028] 2,6-dimethyl-1,4-benzoquinone
[0029] trimethyl-1,4-benzoquinone
[0030] tetramethyl-1,4-benzoquinone
[0031] methoxy-1,4-benzoquinone
[0032] ethoxy-1,4-benzoquinone
[0033] propoxy-1,4-benzoquinone
[0034] 2,5-dimethoxy-1,4-benzoquinone
[0035] 2,6-dimethoxy-1,4-benzoquinone
[0036] trimethoxy-1,4-benzoquinone
[0037] tetramethoxy-1,4-benzoquinone
[0038] hydroxymethyl-1,4-benzoquinone
[0039] hydroxyethyl-1,4-benzoquinone
[0040] hydroxypropyl-1,4-benzoquinone
[0041] 2,5-dihydroxymethyl-1,4-benzoquinone
[0042] 2,6-dihydroxymethyl-1,4-benzoquinone
[0043] tri(hydroxymethyl)-1,4-benzoquinone
[0044] tetra(hydroxymethyl)-1,4-benzoquinone
[0045] phenyl-1,4-benzoquinone
[0046] 2,3-dimethoxy-5-methyl-1,4-benzoquinone, and
[0047] 2-hydroxymethyl-6-methoxy-1,4-benzoquinone.
[0048] An advantage of the substituted benzoquinones as defined
herein is a reduction in oxidation potential compared to
unsubstituted benzoquinone which has an oxidation potential of
approximately +400 mV (versus SCE). It has been shown that addition
of methyl groups to 1,4-benzoquinone results in a negative shift in
redox potential of approxirnately 55 mV per methyl group, due to
the electron-donating nature of methyl groups (Driebergen et al.,
Analytica Chimica Acta, 233 (1990) pp 251-268). This enables
measurements to be performed at lower oxidation potential and
reduces the oxidation of interferent species present in samples
such as ascorbate, urate and paracetamol. Examples of substituted
1,4-benzoquliones with lower oxidation potentials compared to
1,4-benzoquinone are shown in Table 1 below:
1 Compound Solubility (mg/L) E.sub.ox (mV) 1,4-benzoquinone 1.11
E+04 (SRC) 278 2,6-dimethyl-1,4-benzoquinone 7.88 E+03 (SRC) 137
Tetramethyl-1,4-benzoquinone 4.43 E+02 (SRC) -37
Phenyl-1,4-benzoquinone 1.14 E+03 (SRC) 229
2,3-dimethoxy-5-methyl-1,4- 1.11 E+04 (SRC) 156 benzoquinone
2,6-dimethoxy-1,4-benzoquinone 7.06 E+04 (SRC) 166
2-hydroxymethyl-6-methoxy-1,4- 2.85 E+05 (C) 158 benzoquinone SRC =
values obtained from SRC PhysProp Database at
http://esc.syrres.com/interkow/ E.sub.ox = potential of oxidation
peak obtained at a glassy carbon electrode, using pH 6.95 bis-tris
buffer and a.Ag/AgCl/sat. KCl reference electrode. C = calculated
using KowWin from SRC PhysProp Database at
http://esc.syrres.com
[0049] An additional advantage of use of the substituted
benzoquinones is in the ability of these mediator compounds to
oxidize reduced enzyme in place of the physiological electron
acceptor, oxygen. This results in a sensor response that is
independent of the oxygen tension of the sample. This is an
advantage compared to sensors based on the measurement of hydrogen
peroxide production or oxygen depletion caused by the reaction
between enzyme and substrate.
[0050] Suitable benzoquinones for use in the sensor of the
invention may be selected on the basis of their solubility compared
to benzoquinone. The solubility of the substituted benzoquinones
can be reduced by substitution of groups such as alkyl or aryl
groups onto the ring. Solubility of the compound of formula (I) is
an important factor in the proper functioning of the sensor in a
flowing stream. Low solubility in water and aqueous phases is
helpful in providing stability and conveniently, for use in a
flowing stream, the compound of formula (1) should have a
solubility of from 400 mg/L to 10,000 mg/L in pure water.
[0051] Sensors of the invention for use in static solution will
advantageously comprise substituted benzoquinones which have higher
solubility compared to unsubstituted benzoquinone. The solubility
of the substituted benzoquinones can be increased by substitution
of groups such as hydroxy, alkoxy or hydroxyalkyl onto the ring.
Relatively high solubility in water and aqueous phases is helpful
in providing enhanced sensitivity of the sensor response in static
solution. Conveniently in this embodiment, the compound of formula
(I) will have a solubility of at least 10,000 mg/L in pure
water.
[0052] Solubility in common organic solvents is desirable to
facilitate fabrication of the sensors of the present invention, and
conveniently the compound of formula (I) will have a solubility of
at least 20,000 mg/L and preferably higher, in at least one of
methanol, ethanol, propanol, other lower alkanols, chloroform,
dichloromethane or other chlorinated alkanes and acetone and other
low molecular weight solvents.
[0053] Preferably the mediator is specific to reduced enzymes, i.e.
under the conditions of the analysis, the mediator only accepts
electrons from the redox site of a reduced enzyme. In practice it
is likely that this will be the case when operating at the
preferred potential (see below). However, specificity is not
essential and the system may be operated satisfactorily provided
that the mediator is selective for reduced enzyme, i.e. under the
conditions of the analysis the mediator tends to accept electrons
from the redox site of the reduced enzyme in preference to any
other electron donor available to the mediator.
[0054] In one embodiment of the invention, groups R.sup.1, R.sup.2,
R.sup.3 and R.sup.4 may be selected from any alkyl group,
preferably C.sub.1-3 alkyl. The alkyl group includes methyl, ethyl
and propyl and is preferably methyl. Another suitable configuration
has R.sup.1, R.sup.2 and R.sup.3 all hydrogen atoms and group
R.sup.4 a phenyl group. If there is more than one alkyl group or a
phenyl group, these substituents render the substituted
benzoquinone relatively insoluble which provides a mediator of
particular advantage for use in a flowing stream. It is preferred
in that embodiment that the substituted benzoquinones used have a
solubility of not more than 10,000 mgL.sup.-1 in water at room
temperature (25.degree. C.), and not less than 1000 mgL.sup.-1. As
specific examples of useful compounds there may be mentioned
2,6-dimethyl-1,4-benzoquinone, which has a solubility of 7888
mgL.sup.-1 in water at 25.degree. C., tetramethyl-1,4-benzoquinone,
which has a solubility of 443 mgL.sup.-1 in water at 25.degree. C.,
phenyl-1,4-benzoquinone, which has a solubility of 1135 mgL.sup.-1
in water at 25.degree. C., and 2,5-dimethyl-1,4-benzoquinone, which
has a solubility of 7010 mgL.sup.-1 in water at 25.degree. C.,
which may be compared to the parent compound 1,4-benzoquinone which
has a solubility of 11,000 mgL.sup.-1 in water at 18.degree. C.
[0055] An alkyl group or groups may be substituted by one or more
substituents provided that these do not have a detrimental effect
on the activity of the mediator compounds.
[0056] In another embodiment of the invention, groups R.sup.1,
R.sup.2, R.sup.3 or R.sup.4 may be an alkoxy group or a
hydroxyallcyl group, preferably C.sub.1-3 alkoxy or hydroxy
C.sub.13 alkyl. The alkoxy group includes methoxy, ethoxy and
propoxy and is preferably methoxy. The hydroxyalkyl group includes
hydroxymethyl, hydroxyethyl and hydroxypropyl and is preferably
hydroxymethyl. If there is more than one alkoxy group or
hydroxyalkyl group, these substituents render the substituted
benzoquinone relatively soluble which provides mediator of
particular advantage for use in static solution. It is preferred
that in this aspect of the invention, that the substituted
benzoquinones used have a solubility of at least 10,000 mgL.sup.-1
in water at room temperature (25.degree. C.). As specific examples
of useful compounds there may be mentioned
2,6-dimethoxy-1,4-benzoquinone which has a solubility of 70,600
mgL.sup.-1 in water at 25.degree. C.,
2,3-dimethoxy-5-methyl-1,4-benzoqui- none which has a solubility of
11,000 mgL.sup.-1 in water at 25.degree. C. and
2-hydroxymethyl-6-methoxy-1,4-benzoquinone.
[0057] Some of the compounds useful as mediators are known and are
commercially available. Alternatively, they may be made by the
application or adaptation of known techniques.
[0058] The mediator compounds disclosed herein are useful in a
variety of amperometric sensor devices and electrode
configurations. The sensors may be based on a 2 or 3 electrode
system and may be of the disposable (single use) or
reusable/semi-disposable type. The sensors may be used either in a
flowing stream of solution or in static solution, depending on the
relative solubility of the mediator used. In its simplest form the
sensor comprises two electrodes (working and counter) which in use
are contacted with the sample being analysed. One electrode, the
working electrode, is coated with the mediator compound. Mediator
may be applied to the electrode by deposition from a solution of
the mediator in a readily evaporable organic liquid. For mediator
that is fairly soluble in aqueous solution, it may also be applied
to the electrode by deposition from an aqueous solution of the
mediator. When the sensor is being used to determine the
concentration of an analyte such as glucose the mediator is coated
with a suitable enzyme. The enzyme can be immobilised on the
surface of the mediator by conventional techniques such as by use
of a self-sustaining gel layer and/or by use of a retention layer,
which is permeable to the analyte. U.S. Pat. No. 4,711,245
describes in greater detail ways in which the mediator and, when
used, the enzyme may be fixed on the working electrode.
[0059] The electrode substrate is chosen from conventional
materials such as carbon pellets, carbon inks, metallized carbon
and metals (such as platinum or palladium), carbon rods, pencil
leads and carbon rods loaded with metal powder. Conventional
electrode configurations which may be used include those disclosed
in U.S. Pat. No. 4,711,245, U.S. Pat. No. 5,200,051 and U.S. Pat.
No. 5,288,636, incorporated herein by reference. The basic chemical
and electrochemical transformations associated with the present
invention are shown below with reference to the glucose/glucose
oxidase system. Prior to introduction of the sample to be analysed
a potential of about +200 mV (versus Ag/AgCl) is applied to the
sensor electrode. This potential is sufficient to cause oxidation
of the mediator at the working electrode, i.e. conversion of
hydroquinone to the corresponding benzoquinone. When the electrodes
are contacted with the sample to be analysed, the enzyme at the
working electrode acts on the glucose resulting in the formation of
the reduced form of the enzyme. The reaction proceeds as in the
reaction scheme below: 2
[0060] The reduced form of the enzyme reduces the oxidized form of
the mediator as follows: 1 GOD RED + M OX -> GOD OX + M RED
[0061] Then, under the applied potential, the reduced form of the
mediator at the working electrode is converted to the oxidized form
and a diffusion limited current generated. This current can be
measured and correlated to the concentration of analyte in the
sample.
[0062] At the electrode potential involved (+200 mV) there may be
some oxidation of interferants. Typically in blood and plasma
samples these are ascorbate, urate and paracetamol. A diffusion
limiting layer may be applied to the working electrode to extend
the sensor to measurement of higher analyte concentrations. This
layer may also reduce the diffusion of some interferents to the
electrode by acting as a diffusion-limiting barrier. This layer can
also act as an electrostatic barrier to diffusion of interferents
if it contains negatively charged groups. Examples of materials for
use as the diffusion-limiting layer include Nafion.TM. and
cellulose acetate. An effective outer membrane results in a sensor
response which is an accurate reflection of the reduced enzyme
concentration in the sample. The reduced enzyme concentration may
be correlated to analyte concentration.
[0063] It is envisaged that the sensors of the invention will find
most practical utility in the measurement of glucose in blood
samples, although they may be also used for other medical and
non-medical applications, for example in the food industry.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS:
[0064] FIG. 1:
[0065] A sketch of an embodiment of the amperometric sensor of the
invention in the form of a flow cell. The cell consists of a lower
part (A) containing the sensors and an upper part (B) containing
the inlet and outlet for fluid flow. Each sensor is surrounded by a
recess of 0.5 mm width and 0.1 mm depth. The top of the sensor
surface is level with the bottom of the recess. The upper part (B)
contains a rubber O-ring seal and is clamped in place over the
sensors. The parts shown are: sensor 1 (1), sensor 2 (2), Ag/AgCl
reference electrode (3), sensor 3 (4), flow inlet (5), flow outlet
(6), hole for screw (7), O-ring (8) and 0.2 mm recess over sensors
and between inlet and outlet (9).
[0066] FIG. 2A:
[0067] Real, time calibration traces obtained by flow injection
from three 2,6-dimethyl-1,4-benzoquinone mediated glucose sensors
showing the responses to injections of glucose solutions containing
5, 10, 20, 40, 62.5 and 125 mM glucose. The carrier used was
bis-tris saline buffer, pH 6.95.
[0068] FIG. 2B:
[0069] The calibration plots for the three
2,6-dimethyl-1,4-benzoquinone mediated glucose sensor responses
shown in FIG. 2B.
[0070] FIG. 3A:
[0071] Real time calibration traces obtained by flow injection from
three phenyl-1,4-benzoquinone mediated glucose sensors showing the
responses to injections of glucose solutions containing 2.5, 5, 20,
40 and 62.5 mM glucose. The carrier used was bis-tris saline
buffer, pH 6.95.
[0072] FIG. 3B:
[0073] The calibration plots for the three phenyl-1,4-benzoquinone
mediated glucose sensor responses shown in FIG. 3B.
[0074] FIGS. 4A and 4B show the current response and the
corresponding calibration plot in the response of
2,3-dimethoxy-5-methyl-1,4-benzoquino- ne/sarcosine oxidase to
sarcosine.
[0075] FIGS. 5A and 5B show the current response and the
corresponding calibration plot in the response of
2,6-dimethyl-1,4-benzoquinone/sarcosi- ne oxidase to sarcosine.
[0076] FIGS. 6A and 6B show the current response and the
corresponding calibration plot in the response of
2,5-dimethyl-1,4-benzoquinone/sarcosi- ne oxidase to sarcosine.
[0077] FIGS. 7A and 7B show the current response and the
corresponding calibration plot in the response of
2,5-dimethyl-1,4-benzoquinone/glucose oxidase to glucose.
[0078] FIGS. 8A and 8B show the current response and the
corresponding calibration plot in the response of
2,3-dimethoxy-5-methyl-1,4-benzoquino- ne/glucose oxidase to
glucose.
[0079] FIGS. 9A and 9B show the current response and the
corresponding calibration plot in the response of
2-hydroxymethyl-6-methoxy-1,4-benzoqu- inone/glucose oxidase to
glucose.
EXAMPLES
Example 1
Electrode Construction
[0080] The amperometric flow cell was supplied by Drew Scientific,
Cumbria, U.K. FIG. 1 shows a sketch of the flow cell, including the
sensors. The cell was made of either PVC or nylon. Carbon pellets
(2 mm diameter and 4 mm length) were press fitted into the holes in
the lower part (A) so that the front face was flush with the bottom
of the recess. Electrical contact was made to the back face of the
pellets. One of the carbon pellets was modified with Ag/AgCl paste
to act as both a pseudo-reference electrode and counter
electrode.
Example 2
2,6-dimethyl-1,4-benzoquinone Mediated Glucose Sensor
[0081] Unmodified carbon pellets, inserted into the bottom part of
the flow cell as described in Example 1, were prepared as glucose
sensors by successively depositing a mediator layer, an enzyme
layer and an outer diffusion limitation layer. 2-4 .mu.L of 0.5 M
2,6-dimethyl-1,4-benzoquin- one solution in acetone was deposited
into the recess surrounding each pellet and allowed to dry for 3
minutes. A solution containing a mixture of glucose oxidase GOX
(2-4 U/.mu.L), PVA (0.09%) and Nafion (0.31%) was prepared and a
total of 4 .mu.L of the solution was deposited on top of the
benzoquinone layer of each sensor to form the enzyme sensing layer.
The sensors were air dried for 20 minutes before a total of 3 .mu.L
of 2.5% Nafion solution was deposited onto each sensor. The sensors
were air dried for at least 4 hr before use.
Example 3
Use of 2,6-dimethyl-1,4-benzoquinone Glucose Sensor
[0082] Flow injection chronoamperometry was used to show the
activity of these sensors to glucose. A two electrode system was
used, with the three sensors of Example 2 as the working
electrodes, and the pellet modified with silver/silver chloride
acting as the reference and counter electrode. An AutoLab (Eco
Chemie B.V.) electrochemical system was used for the measurements.
The carrier buffer was pH 6.95 bis-tris buffer (40 mM), containing
142 mM NaCl, 0.8 mM Na.sub.2EDTA and 4.2 mM KCl. The woridng
electrodes were poised at a potential of .+-.200 mV versus the
silver/silver chloride reference electrode. FIG. 2A shows anodic
current peaks corresponding to injections of glucose. Calibration
plots resulting from the amperometry data in FIG. 2A are shown in
FIG. 2B. The linear range of response is from 0 to 60 mM
glucose.
Example 4
Phenyl-1,4-benzoquinone Mediated Glucose Sensor
[0083] Unmodified carbon pellets, inserted into the bottom part of
the flow cell as described in Example 1, were prepared as glucose
sensors in an identical manner to the sensors in Example 2, with
the exception of the use of 0.5 M phenyl-1,4-benzoquinone solution
in place of 2,6-dimethyl-1,4-benzoquinone solution.
Example 5
Use of phenyl-1,4-benzoguinone Glucose Sensor
[0084] Flow injection chronoamperometry was used to demonstrate the
response of the sensors in Example 4 to glucose, using the same
experimental methodology given in Example 3. FIG. 3A shows anodic
current peaks corresponding to injections of glucose. Calibration
plots resulting from the amperometry data in FIG. 3A are shown in
FIG. 3B. The linear range of response is from 0 to 60 mM
glucose.
Example 6
[0085] Chronoamperometry was used to show the mediator ability of
some compounds in solution to sarcosine oxidase. The change in
current at a glassy carbon electrode was observed on consecutive
addition of aliquots of 1 M sarcosine stock solution to buffer (pH
7.4) containing 100 units/ml sarcosine oxidase and either 0.2 M
2,6-dimethyl-1,4-benzoquinone- , 0.2 M
2,5-dimethyl-1,4-benzoquinone or a saturated solution of
2,3-dimethoxy-5-methyl-1,4-benzoquinone.
[0086] The electrode potential was held at +250 mV vs.
Ag/AgCl/sat/KCl and platinum counter electrode was used.
[0087] FIG. 4A shows the current response on the addition of
sarcosine to a solution containing
2,3-dimethoxy-5-methyl-1,4-benzoquinone and sarcosine oxidase. FIG.
4B shows the corresponding calibration plot. FIG. 5A shows the
current response on the addition of sarcosine to a solution
containing 2,6-dimethyl-1,4-benzoquinone and sarcosine oxidase, and
FIG. 5B shows the corresponding calibration plot. FIG. 6A shows the
current response of 2,5-dimethyl-1,4-benzoquinone/sarcosine oxidase
to sarcosine, and FIG. 6B shows the corresponding calibration
plot.
Example 7
[0088] Chronoamperometry was used to show the mediator ability of
some compounds in solution to glucose oxidase. The change in
current at a glassy carbon electrode was observed on consecutive
addition of aliquots of 1 M glucose stock solution to buffer (pH
7.4) containing 100 units/ml glucose oxidase and either 0.2 M
2,5-dimethyl-1,4-benzoquinone, a saturated solution of
2,3-dimethoxy-5-methyl-1,4-benzoquinone or a saturated solution of
2-hydroxymethyl-6-methoxy-1,4-benzoquinone.
[0089] The electrode potential was held at +250 mV vs.
Ag/AgCl/sat/KCl and platinum counter electrode was used.
[0090] FIG. 7A shows the current response of
2,5-dimethyl-1,4-benzoquinone- /glucose oxidase to glucose, and
FIG. 7B shows the corresponding calibration plot. FIG. 8A shows the
current response of 2,3-dimethoxy-5-methyl-1,4-benzoquinone/glucose
oxidase to glucose and FIG. 8B shows the corresponding calibration
plot. FIG. 9A shows the current response of
2-hydroxymethyl-6-methoxy-1,4-benzoquinone/glucose oxidase to
glucose, and FIG. 9B shows the corresponding calibration plot.
* * * * *
References